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NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.na u e.com 57
Ch oma in-cell s imula ion igge s as
millimola mi ochond ial Ca2+ ansien s
ha modula e sec e ion
May e Mon e o*, Ma ia Te esa Alonso*, Es ela Ca nice o*, Inmaculada Cuchillo-Ibáñez†, Almudena Albillos†,
An onio G. Ga cía†, Ja ie Ga cía-Sancho* and Ja ie Al a ez*‡
*Ins i u o de Biología y Gené ica Molecula (IBGM), Uni e sidad de Valladolid y Consejo Supe io de In es igaciones Cien í icas (CSIC), Depa amen o de Bioquímica
y Biología Molecula y Fisiología, Facul ad de Medicina, Ramón y Cajal 7, E-47005 Valladolid, Spain
†Ins i u o de Fa macología Teó ilo He nando, Depa amen o de Fa macología y Te apéu ica, Facul ad de Medicina, Uni e sidad Au ónoma de Mad id, A zobispo
Mo cillo 4, E-28029 Mad id, Spain
‡e-mail: jal a [email protected] a.es
Ac i a ion o calcium-ion (Ca2+) channels on he plasma memb ane and on in acellula Ca2+ s o es, such as he
endoplasmic e iculum, gene a es local ansien inc eases in he cy osolic Ca2+ concen a ion ha induce Ca2+ up ake
by neighbou ing mi ochond ia. He e, by using mi ochond ially a ge ed aequo in p o eins wi h di e en Ca2+ a ini ies,
we show ha hal o he ch oma in-cell mi ochond ia exhibi su p isingly apid millimola Ca2+ ansien s upon
s imula ion o cells wi h ace ylcholine, ca eine o high concen a ions o po assium ions. Ou esul s show a igh
unc ional coupling o ol age-dependen Ca2+ channels on he plasma memb ane, yanodine ecep o s on he
endoplasmic e iculum, and mi ochond ia. Cell s imula ion gene a es localized Ca2+ ansien s, wi h Ca2+ concen a ions
abo e 20–40
µM, a hese unc ional uni s. P o onopho es abolish mi ochond ial Ca2+ up ake and inc ease s imula ed
sec e ion o ca echolamines by h ee- o i e old. These esul s indica e ha mi ochond ia modula e sec e ion by
con olling he a ailabili y o Ca2+ o exocy osis.
u ing cell ac i a ion, some mi ochond ia ake up Ca2+ om
cy osolic mic odomains o high Ca2+ concen a ion ha a e
gene a ed by ac i a ion o nea by Ca2+ channels1–3. Changes
in he mi ochond ial Ca2+ concen a ion ([Ca2+]M) a e he e o e
he e ogeneous a he subcellula le el, and he [Ca2+]M ansien in
a pa icula mi ochond ion depends on i s p oximi y o a Ca2+
sou ce1–3. Inc eases in [Ca2+]M ac i a e dehyd ogenases2,4, coupling
ATP p oduc ion o ene gy demands. In addi ion, inc easing
amoun s o e idence indica e ha mi ochond ia may also modu-
la e homeos asis o he cy osolic Ca2+ concen a ion ([Ca2+]c). Fo
example, in neu ons and ch oma in cells, mi ochond ia ac as
apid and e e sible Ca2+ bu e s du ing cell s imula ion5–9 and in
he clea ance o la ge Ca2+ loads10. Mo eo e , o e loading o mi o-
chond ia wi h Ca2+ may be in ol ed in he de elopmen o se e al
pa hological condi ions, including ischaemia- epe usion lesions,
neu o oxici y and neu odegene a i e diseases, whe e ATP deple-
ion, o e p oduc ion o oxida i e adicals and elease o apop o ic
ac o s lead o cell damage11–14. Howe e , [Ca2+]M measu emen s
du ing cell s imula ion, ob ained by a a ie y o di e en ech-
niques, ha e p o ided alues only in he low mic omola ange1–4,9.
Al hough hese changes a e enough o ac i a e mi ochond ial
me abolism, hei physiological signi icance o cellula Ca2+
homeos asis emains unclea . Simila ly, he ac ual ele ance o
mi ochond ial Ca2+ o e load is obscu e. We show he e ha [Ca2+]M
can in ac each he millimola ange du ing s imula ion o ch o-
ma in cells, and ha his la ge mi ochond ial Ca2+ up ake egu-
la es he a ailabili y o Ca2+ o he sec e o y machine y.
Resul s
We used h ee ypes o mi ochond ially a ge ed aequo in wi h di e -
en Ca2+ sensi i i ies. This allowed us o measu e [Ca2+]M a a wide ange
o concen a ions, om he submic omola o he millimola . Wild-
ype aequo in econs i u ed wi h na i e coelen e azine (AEQ1) shows
a ini y o Ca2+ in he concen a ion ange 0.3–8
µM; wild- ype aequo-
in econs i u ed wi h coelen e azine n (AEQ2) co e s he ange 1–40
µM Ca2+; and mu a ed low-Ca2+-a ini y aequo in15 econs i u ed wi h
coelen e azine n (AEQ3) can measu e Ca2+ in he concen a ion ange
20
µM o 1
mM16. A u he dec ease in he Ca2+ sensi i i y o aequo in
was ob ained by using AEQ3 a 22
°C ( e . 17). Ano he p ope y o
aequo in, i s i e e sible consump ion as i emi s ligh , enables i o be
used in de ec ing subcellula he e ogenei ies in [Ca2+] ( e . 18). Aequo-
in wi h high Ca2+ a ini y is apidly consumed in a eas o high [Ca2+].
A e ha , measu emen s e lec only he beha iou o low-[Ca2+]
a eas. Ins ead, when low-Ca2+-a ini y aequo in is used, only egions
wi h high [Ca2+] con ibu e signi ican ly o he luminescence. In each
case, he ela i e ac ion o aequo in consumed p o ides an es ima ion
o he size o he di e en [Ca2+] pools.
He e ogenei y o he [Ca2+]M esponse. Wi h ega d o hei
esponse o [Ca2+], we ound wo main popula ions o mi ochond ia
in ch oma in cells. Figu e 1a, b show simila expe imen s pe o med
wi h ei he AEQ1 o AEQ2. Cell depola iza ion using a medium wi h
a high po assium-ion (K+) concen a ion igge ed a apid consump-
ion o 40–50% aequo in in bo h cases (do ed line). Calib a ed in
[Ca2+] (con inuous line), we es ima ed peaks o 5
µM and 30
µM Ca2+
when using AEQ1 and AEQ2, espec i ely. Subsequen depola iza-
ions p oduced smalle [Ca2+]M peaks o simila magni ude (abou 2
µM) when calcula ed using bo h ypes o aequo in. Figu e 1c, d show
simila expe imen s bu using ca eine ins ead o high [K+]. Ca eine
ac i a es yanodine ecep o s (Ca2+ channels ound on he endoplas-
mic e iculum), inducing Ca2+ elease om he endoplasmic
e iculum19. The i s addi ion o ca eine esul ed in he consump-
ion o ~30% o AEQ1 o AEQ2, a alue ha con e s o es ima ed
[Ca2+]M peaks o 5
µM and 40
µM, espec i ely. Subsequen addi ions
o ca eine p oduced much smalle [Ca2+]M peaks ha ga e simila
calib a ed esul s wi h bo h aequo ins ([Ca2+]M 2
µM). The [Ca2+]c
esponse o he same pa e n o s imuli was e y di e en , wi h con-
secu i e addi ions o high K+ concen a ions o ca eine p oducing
simila [Ca2+]c peaks19. The disc epancy can be explained only i
~50% o ch oma in-cell mi ochond ia espond o s imula ion wi h
2
µM [Ca2+]M peaks while he es unde go a much la ge inc ease in
[Ca2+]M. Aequo in in he la e pool o mi ochond ia is consumed
du ing he i s s imulus, and subsequen s imuli e oke only he
smalle esponse om he o he mi ochond ia. In con as o HeLa
D
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58 NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.na u e.com
cells, o which ex ensi e mi ochond ial lumenal di usion has been
epo ed20, la ge [Ca2+]M peaks we e no es o ed in ch oma in cells
a e 20
min wi hou s imula ion (Fig. 1).
Co-localiza ion o Ca2+-en y and - elease si es. We used he pa -
e n o AEQ2 consump ion du ing he consecu i e addi ion o high
K+ le els and ca eine o s udy he deg ee o o e lapping among he
mi ochond ial pools ha espond o each s imulus wi h la ge
[Ca2+]M peaks. Ca eine was unable o induce any la ge [Ca2+]M
esponse a e ea men o cells wi h high [K+]; howe e , ca eine
applied be o e K+ esul ed in he consump ion o ~60% o he aequ-
o in ha was able o espond o high [K+] wi h la ge [Ca2+]M peaks
(Fig. 1e, ). I we conside he amoun o AEQ2 consump ion a e
wo consecu i e pulses o high [K+] and ca eine o be 100%, hen
he ini ial pulse o high [K+] esul ed in he consump ion o 96.5
±
0.4% (mean
±
s.e.m.; n
=
10) o his aequo in. Howe e , i ca eine
we e added be o e K+, he ini ial ca eine pulse esul ed in he con-
sump ion o only 59
±
2% o AEQ2 (n
=
16). We conclude ha abou
30% o mi ochond ia co-localize wi h bo h plasma-memb ane Ca2+
channels (which a e ac i a ed by high [K+]) and yanodine ecep-
o s (which a e ac i a ed by ca eine). An addi ional 20% o mi o-
chond ia co-localize wi h plasma-memb ane Ca2+ channels, bu no
wi h yanodine ecep o s. The emaining 50% o he mi ochond ia
a e appa en ly loca ed a away om hese Ca2+ ho spo s.
Millimola [Ca2+]M ansien s. Peak [Ca2+]M alues calcula ed using
AEQ2 (30–40
µM) a e unde es ima ed because he aequo in is con-
sumed. Quan i ica ion o he eal alues o he la ge [Ca2+]M peaks
equi ed he use o he lowe -Ca2+-a ini y AEQ3. Figu e 2a shows he
[Ca2+]M changes induced by a se ies o high-[K+] pulses. The ini ial
[Ca2+]M peak eached 200
µM, and subsequen peaks dec eased in
magni ude as soon as aequo in consump ion app oached 50–60%.
These alues a e s ill unde es ima es, because la ge [Ca2+]M changes
ake place in only hal o he mi ochond ia. Taking his ac in o
accoun (Fig. 2b), we es ima e [Ca2+]M peaks o simila magni ude o
equal epe i i e s imuli, wi h peaks being ~300
µM (340
±
20
µM, n
=
15) o 10-s pulses and ~700
µM (670
±
30
µM, n
=
11) o 30-s pulses.
Figu e 2c shows he e ec o ca eine, which p oduced a i s [Ca2+]M
peak o 200
µM and hen dec easing peaks as consump ion
app oached 40%. Again, i da a a e ecalib a ed conside ing only he
ac i e mi ochond ial space (Fig. 2d), we ob ain a se ies o simila as
[Ca2+]M ansien s eaching peak alues o nea 500
µM (430
±
30
µM,
n
=
8). The e ec o ca eine esul ed om Ca2+ elease om in acellu-
la s o es, because i p oduced he same [Ca2+]M ansien in Ca2+- ee
medium (da a no shown).The physiological agonis ace ylcholine
also p oduced a la ge [Ca2+]M peak (280
±
50
µM, n
=
4; Fig. 2e). Finally,
he high-[K+]-induced [Ca2+]M ansien was e e sibly abolished by
he p o onopho e ca bonyl cyanide p- i luo ome hoxy phenyl
hyd azone (FCCP) (Fig. 2 ).
Re u n o [Ca2+]M o es ing le els a e s imula ion was as ,
eaching backg ound alues wi hin 1–2 min. CGP37157, an inhibi-
o o he mi ochond ial Na+/Ca2+ exchange , educed he a e o
[Ca2+]M dec ease by 60% (hal - ime 62
±
3 s, n
=
6; con ol, hal - ime
25
±
1
s, n
=
9; Fig. 3a). The [Ca2+]M and [Ca2+]c ansien s induced by
high [K+] ollow e y simila ime cou ses (Fig. 3b). This means ha
[Ca2+]M is able o ollow apid oscilla ions in [Ca2+]c, bu a concen-
a ions mo e han wo o de s o magni ude highe . Ca2+ exi om
mi ochond ia and Ca2+ up ake by mi ochond ia di e ed in sensi i -
i y o empe a u e. The a e o [Ca2+]M inc ease was e y simila a
22
°C (67
±
2
µM
s–1, n
=
17) and 37
°C (58
±
3
µM
s–1, n
=
15). The
Figu e 1 E ec s o high K+ concen a ions o ca eine on [Ca2+]M, measu ed
wi h AEQ1 o AEQ2. The e ec s o 10-s pulses wi h medium con aining 70
mM K+
(K+) and/o 30-s pulses wi h 50
mM ca eine (C) on [Ca2+]M (con inuous line) and he
aequo in consump ion ( alues shown a e he pe cen age o aequo in consumed;
do ed line), measu ed ei he wi h AEQ1 (a, c) o wi h AEQ2 (b, d– ). The o al amoun
o aequo in consumed was measu ed a he end o e e y expe imen by pe usion o
100
µM digi onin plus 10 mM Ca2+. The b eaks in a–d indica e 15
min wi hou
s imula ion. The empe a u e was 37
°C.
aAEQ1
5 min
0
1
2
3
4
5
0
20
40
60
80
100
K+
K+
K+K+K+K+K+
0
10
20
30 5 min
K+
K+K+K+K+K+K+
0
20
40
60
80
100
bAEQ2
2
4
65 min
0
20
40
60
80
100
[Ca ] (µM)
2+ M
Pe cen age consumed
C
CCCCCC
5 min
00
20
40
60
80
100
[Ca ] (µM)
2+ M
Pe cen age consumed
10
20
30
40 C
CCCC CC
00
20
40
60
80
100
5 min
CCC
10
20
30
K+K+
C
C
0
10
20
30
0
20
40
60
80
100
5 min
K+CK+
K+
cd
e
AEQ1 AEQ2
AEQ2 AEQ2
K+
Figu e 2 E ec s o high K+ concen a ions o ca eine on [Ca2+]M measu ed
wi h AEQ3. a, c, The e ec s o 10-s o 30-s pulses wi h a high K+ concen a ion (K+)
o 30-s pulses wi h 50
mM ca eine (C) on [Ca2+]M, measu ed wi h AEQ3 (con inuous
line), and on he pe cen age o aequo in consump ion (do ed line). b, d, Resul s o
expe imen s ha we e he same as hose in a, c bu ecalib a ed by assuming ha
[Ca2+]M changes occu ed in only 58% and 48%, espec i ely, o he o al space. e, The
e ec o a 10-s pulse wi h 10 mM ace ylcholine (ACh) ollowed by 30-s K+ pulses. , 2
µM FCCP and 10-s K+ pulses we e pe used as indica ed. Tempe a u e was 37
°C.
CCC
CCCC
C
CCCC
a
c
e
b
d
5 min
5 min
5 min 5 min
5 min
5 min
0
50
100
150
200
0
20
40
60
80
100
K+
K+
K+
K+
K+
K+
00
50
100
100
80
60
40
20
150
200
250
[Ca ] (µM)
2+ M
0
100
200
300
400
500
[Ca ] (µM)
2+ M
0
200
400
600
800
10 s
K+
10 s
0
200
400
600
ACh
0
200
100
300
400
K+
30 s K+
30 s K+
30 s
K+
30 s K+
10 s
K+
10 s
FCCP
K+
10 s
K+
10 s
K+
10 s K+
10 s
10 s
10 s
30 s
30 s
K+
30 s K+
30 sK+
30 s
30 s
Pe cen age consumed
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kine ics o he [Ca2+]c ansien we e also simila a bo h empe a-
u es (Fig. 3c). Howe e , he a e o Ca2+ exi om mi ochond ia
was nea ly ou old slowe a 22
°C han a 37
°C (hal - imes 92
±
4
s,
n
=
11, and 25
±
1
s, n
=
9, a 22
°C and 37
°C, espec i ely). The e o e,
a 22
°C, he [Ca2+]M ansien las ed o much longe han he
cy osolic one. The heigh o he [Ca2+]M peaks induced by he s im-
uli was also la ge a 22
°C han a 37
°C (10-s high-[K+] pulse,
[Ca2+]M peak
=
550
±
30
µM, n
=
10; 30-s high-[K+] pulse, [Ca2+]M
peak = 870
±
30
µM, n
=
8; ca eine, [Ca2+]M peak
=
520
±
50
µM, n
=
4;
ace ylcholine, [Ca2+]M peak
=
670
±
40
µM, n
=
6). These indings a e
consis en wi h he epo ed abili y o mi ochond ia o p olong he
[Ca2+]c peaks by seques e ing Ca2+ du ing he s imula ion and
eleasing i a e wa ds, in expe imen s pe o med a oom
empe a u e5–9. Ou esul s indica e ha he abili y o mi ochond ia
o p olong [Ca2+]c ansien s should be much less a 37
°C. Figu e 3c
also shows he inhibi ion by CGP37157 o mi ochond ial Ca2+
elease a 22
°C (hal - ime 380
±
60s, n
=
3; 76% inhibi ion).
Mi ochond ia as biosenso s o local [Ca2+]c. The la ge [Ca2+]M
peaks ha we ha e obse ed can be gene a ed only i mi ochond ia
a e exposed o e y high cy osolic Ca2+ concen a ions. As he a e o
Ca2+ up ake by mi ochond ia is p opo ional o [Ca2+]c ( e s 1, 10, 21,
22), we can es ima e he size o he local [Ca2+]c ansien s by compa -
ing he a es o [Ca2+]M inc ease obse ed in in ac cells wi h hose
ob ained in pe meabilized cells pe used wi h known Ca2+ concen a-
ions (Fig. 4a). A simila app oach has been used in pe meabilized
RBL mucosal mas cells o show ha inosi ol-1,4,5- isphospha e
p oduces local [Ca2+]c ansien s abo e 16
µM ( e . 21). We ound
ha he consump ion o aequo in by pe meabilized cells exposed o
Ca2+ bu e s ollowed a mono onic a e, as would be expec ed o a
single mi ochond ial pool wi h a homogeneous a e o Ca2+ up ake.
This esul indica es ha he he e ogeneous esponses shown in Fig.
1 a e due o he subcellula localiza ion o each mi ochond ial pool
and no o di e ences in Ca2+-up ake p ope ies. The ela ionship
be ween he a e o [Ca2+]M inc ease and [Ca2+]c is plo ed in Fig. 4b.
The a e o [Ca2+]M inc ease was negligible below 4 µM [Ca2+]c, bu
inc eased s eeply abo e hese alues and was ully blocked by u he-
nium ed, a blocke o he mi ochond ial Ca2+ unipo e . Fi ed al-
ues o he maximal a e o up ake and o he [Ca2+]c ha gi es a hal -
maximal up ake a e we e 225
µM
s–1 and 43
µM, espec i ely. This KM
alue is consis en wi h p e ious measu emen s made in ch oma in
cells10,22. The a e o inc ease o [Ca2+]M ob ained in cells s imula ed
wi h high [K+] co esponded o ~20
µM [Ca2+]c. The a es o [Ca2+]M
inc ease obse ed in in ac cells s imula ed wi h ca eine o ace ylcho-
line we e, espec i ely, 110
±
9
µM
s–1 (n
=
11) and 94
±
7
µM
s–1 (n
=
6),
which co espond o cy osolic Ca2+ concen a ions o 40
µM and 30
µM, espec i ely.
In hose mi ochond ia unde going small (1–2 µM) [Ca2+]M an-
sien s on s imula ion (Fig. 1), he a e o high-[K+]-induced [Ca2+]M
inc ease was only 0.29
±
0.02
µM
s–1 (n
=
35). This a e co esponds
o a [Ca2+]c o 2–3 µM, close o he mean [Ca2+]c alues measu ed
using Fu a-2 (Fig. 3b).
Figu e 3 E ec s o CGP37157, an inhibi o o he mi ochond ial Na+/Ca2+
exchange , and empe a u e on he a e o [Ca2+]M dec ease a e
s imula ion. a, Con ol cells and cells incuba ed a 37
°C o 2
min wi h 20
µM
CGP37157 (CGP) we e s imula ed wi h a 10-s pulse o high [K+] (K+). The inhibi o
was also p esen a e he s imulus. b, Compa ison o he kine ics o he [Ca2+]M
(con inuous line) and [Ca2+]c (do ed line) ansien s a 37
°C a e s imula ion wi h a
10-s pulse o high [K+]. c, 10-s pulses o high [K+] we e gi en a 22
°C in he p esence
o absence o 20 µM CGP37157, as indica ed. The kine ics o he [Ca2+]c ansien
a 22
°C is also shown (do ed line). Expe imen s we e pe o med using AEQ3 and
calib a ed using he assump ion ha [Ca2+]M changes occu ed in only 55% o he
o al space.
2 min
[Ca ] (µM)
2+ M[Ca ] (µM)
2+ M
[Ca ] (µM)
2+ M
[Ca ] (µM)
2+ M
800
400
300
200
100
0Con ol
22 C
600
400
200
0K+
K+
K+
K+
400
300
200
100
0
0
0.5
1
1.5
2
CGP
CGP
[Ca ] (µM)
2+ c
0
0.5
1
1.5
2
o
37 C
o
ab
c
Figu e 4 Dependence o he a e o mi ochond ial Ca2+ up ake on [Ca2+]c.
a, An inc ease in [Ca2+]M was induced by pe usion o pe meabilized cells wi h bu e s
o known Ca2+ concen a ion, as indica ed. In he cu e labelled ‘20
µM+RR’, he
bu e con aining 20
µM Ca2+ was added in he p esence o 4 µM u henium ed.
These expe imen s we e ca ied ou using AEQ3 a 22
°C, o inc ease he sensi i i y
o he measu emen s a high up ake a es. b, Plo o he maximum a es o up ake
ob ained a e e y Ca2+ concen a ion. Da a a 2
µM and 4
µM [Ca2+]c ( a es o up ake
0.2 and 0.45
µM
s–1, espec i ely) we e ob ained using AEQ2. E o ba s show s.e.m.
o ou de e mina ions made o each poin .
0 20 40 60 80 100
0
50
100
150
200
a
b
0
500
1,000
1,500
60 µM
Up ake a e (µM s )
–1 [Ca ] (µM)
2+ M
[Ca ] (µM)
2+ c
100 µM
40 µM
20 µM 10 µM
7 µM
20 µM + RR
10 s
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Modula ion o sec e ion by mi ochond ia. We analysed he physi-
ological ele ance o mi ochond ial Ca2+ up ake by compa ing ca e-
cholamine sec e ion induced by s imula ion wi h high [K+], ca eine
o ace ylcholine, bo h in he p esence and in he absence o he p o -
onopho e ca bonyl cyanide m-chlo ophenyl-hyd azone (CCCP).
We expec ed ha CCCP, by p e en ing Ca2+ seques a ion by mi o-
chond ia, would allow a la ge local inc ease in subplasmalemmal
[Ca2+]c and hence po en ia e sec e ion. This was he case (Fig. 5). In
esponse o cellula challenge wi h ace ylcholine (100
µM o 2 s, a 2-
min in e als), ai ly ep oducible sec e o y peaks o a ound 300
nA
we e p oduced; CCCP (2
µM) e e sibly inc eased he sec e o y
esponse o ~1,000
nA (Fig. 5a). Simila esul s we e ob ained when
cells we e challenged wi h high [K+] (70
mM K+, 2
s; Fig. 5b) o ca -
eine (50
mM, 2
s; Fig. 5c). Figu e 5d shows he a e aged o al sec e-
ion ob ained om en epe i ions o each o hese expe imen s. The
small e ec on sec e ion o CCCP alone disappea ed when he in e -
al be ween s imuli was inc eased o 5
min, bu po en ia ion o sec e-
ion emained unchanged.
Po en ia ion o sec e ion by CCCP was no due o inc eased Ca2+
up ake om ex acellula sou ces. Expe imen s in which he up ake
o 45Ca2+ was measu ed showed ha CCCP did no inc ease he ne
Ca2+ up ake induced by any o he s imuli (da a no shown). Nei he
was he e ec o CCCP media ed by a possible Ca2+ elease om he
endoplasmic e iculum. When cells we e ea ed wi h a combina-
ion o 10
mM ca eine, 10
µM yanodine and 1
µM hapsiga gin in
Ca2+- ee medium o 30 min, ca eine p oduced no sec e o y
esponse, bu he po en ia ion by CCCP o K+-induced ca echo-
lamine sec e ion was unmodi ied (da a no shown). Finally, mi o-
chond ial inhibi o s p oduced he same e ec s as CCCP. We
s imula ed cells wi h 2-s high-[K+] pulses e e y 2
min. Oligomycin
(3
µM, p eincuba ion 90
s), which blocks ATP p oduc ion bu
should no dec ease he mi ochond ial memb ane po en ial, had
li le e ec on K+-s imula ed sec e ion. The mean no malized peak
sec e ion was 100
±
12% in con ol pulses, and 139
±
21% in he
p esence o oligomycin (mean
±
s.e.m, n
=
11; P>0.1, S uden ’s -
es ). Howe e , a combina ion o 4
µM o enone (which blocks es-
pi a ion) and 3
µM oligomycin p oduced a la ge po en ia ion o
high-K+-induced sec e ion. The mean no malized peak sec e ion in
con ol pulses was 100
±
6%, and i inc eased o 204
±
21% (n
=
11;
P<0.001, - es ) in he p esence o oligomycin plus o enone.
Discussion
I was as onishing o ind ha some mi ochond ia can unde go, in
s imula ed in ac cells, apid e e sible inc eases in Ca2+ concen a-
ion om he submic omola o he millimola ange. Such high
mi ochond ial Ca2+ concen a ions we e p e iously associa ed only
wi h pa hological phenomena ela ed o cell damage o he ini ial
s ages o apop osis10–13. These [Ca2+]M alues con as wi h hose
ob ained in a simila p epa a ion using he luo escen dye hod2
( e . 9). The disc epancy can be explained by di e ences in he spe-
ci ici y o a ge ing and ange o measu emen o he p obes.
Co-localiza ion o hose mi ochond ia unde going la ge [Ca2+]M
ansien s wi h bo h plasma-memb ane Ca2+ channels and yanodine
ecep o s e eals he p esence o unc ional uni s o Ca2+-induced
Figu e 5 CCCP enhances sec e o y esponses e oked by ace ylcholine, high
K+ concen a ion o ca eine. a–c, Cells we e supe used wi h K ebs–HEPES
medium. A e a 10-min equilib a ion pe iod, o ge a s able baseline sec e ion, cells
we e challenged a 2-min in e als wi h 2-s pulses o a, 100
µM ace ylcholine (ACh),
b, medium wi h a high K+ concen a ion (70 mM, wi h iso-osmo ic educ ion o Na+),
o c, ca eine (50 mM). The do s a he bo om o he panels each ep esen one
challenge wi h one o hese sec e agogues. In addi ion, 2
µM CCCP was applied in
wo phases, each o 90
s, as indica ed by he ba s. G aphs ep esen o iginal aces
om h ee ypical expe imen s using pulses o ace ylcholine (a), high [K+] (b) o
ca eine (c). The ampli udes o sec e ion peaks a e exp essed as oxida ion cu en s
(measu ed in nA). d, A e aged esul s ob ained om en epe i ions each o he
expe imen s desc ibed in a–c. To al sec e ion was calcula ed by in eg a ion o he
a eas o he sec e o y peaks, and is exp essed as µC (µA × s).
a
CCCP CCCP
200 nA
4 min
ACh
b
120 nA
High K
+
High K
+
d
Sec e ion (µC)
0
2
4
6
8
1210
864
20
ACh
Ca eine
CCCP CCCP
Peak numbe
c
Ca eine
CCCP CCCP
4 min
120 nA
4 min CCCP CCCP
Figu e 6 The complex unc ional uni esponsible o he gene a ion o local
high [Ca2+]c ansien s and ca echolamine sec e ion in a bo ine ch oma in
cell. The ol age-dependen Ca2+ channel (VDCC), he Ca2+ unipo e (U) o he uppe
mi ochond ia, he yanodine ecep o (RyR) om he endoplasmic e iculum (ER) and
he sec e o y esicle a e all s a egically loca ed close oge he benea h he plasma
memb ane. Ca2+ en y h ough VDCCs igge s Ca2+-induced Ca2+- elease om he
ER h ough RyRs and gene a es a local [Ca2+]c ho spo o abou 50
µM. In he es o
he cell, he [Ca2+]c inc ease is much less, he esul ing [Ca2+]c being 1–2 µM.
Mi ochond ia ake up Ca2+ h ough he unipo e , wi h up ake depending on he
su ounding [Ca2+]c. The unipo e loca ed close o he [Ca2+]c ho spo in his diag am
will ake up Ca2+ e y quickly and he mi ochond ial [Ca2+] will each nea millimola
le els. This up ake e ec i ely educes he amoun o Ca2+ a ailable o he sec e o y
esicle, allowing a igh con ol o local [Ca2+]c and hence o he sec e o y esponse.
Ca2+ is hen eleased h ough he Na+/Ca2+ exchange . The o he mi ochond ion,
placed a away om he egion o high [Ca2+]c, inc eases i s [Ca2+]M o only abou 2
µM. [Ca2+]ER, he ER Ca2+ concen a ion; [Ca2+]o, he ex acellula Ca2+ concen a ion.
VDCC
Sec e o y
esicle
RyR
Endoplasmic
Re iculum
50 µM
Mi ochond ion
[Ca ]
2+
2+
M
[Ca ]
2+ o
[Ca ]
2+ c
1 µM
[Ca ]
2+ c
500 µM
[Ca ]
2+ M
2 µM
[Ca ]
2+ ER
500 µM
1,000 µM
U
U
~
Ca
Na+
2+
Ca Na+
~
~
© 1999 Macmillan Magazines L d
a icles
NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.na u e.com 61
Ca2+ elease (CICR). Ou da a indica e ha mos o he yanodine
ecep o s in ch oma in cells a e ei he loca ed wi hin hese CICR
unc ional uni s o ha e no mi ochond ia placed nea hem. A p ecise
spa ial ela ionship be ween yanodine ecep o s and mi ochond ia
occu s only a hese unc ional CICR uni s. The la ge size o he mi o-
chond ial pool close o he Ca2+-en y and - elease si es (50%) indi-
ca ed ha i migh be impo an o apid bu e ing o Ca2+ in local
subplasmalemmal egions. We con i med his by showing ha ca e-
cholamine sec e ion induced by se e al s imuli depended ma kedly
on mi ochond ial Ca2+ up ake. When his up ake was blocked, ei he
by collapsing he mi ochond ial ansmemb ane H+ g adien wi h
CCCP o by blocking espi a ion wi h o enone and p e en ing
e e sal o he mi ochond ial ATPase wi h oligomycin, he sec e o y
esponse inc eased se e al- old. These esul s indica e ha mi o-
chond ial Ca2+ up ake con ols he inc ease o [Ca2+]c a exocy ic si es.
In addi ion, blockade o mi ochond ial Ca2+ up ake could allow
sp eading o he Ca2+ wa e o deepe cy osolic a eas, hus a ou ing
mobiliza ion o he ese e pool o sec e o y esicles23. In any case,
ou esul s suppo he idea ha ch oma in cells possess complex
unc ional uni s (Fig. 6) ha clus e oge he all he elemen s needed
o con ol he subplasmalemmal [Ca2+]c ansien s nea exocy ic si es.
The in e play be ween Ca2+-channel ac i a ion, mi ochond ial Ca2+
up ake and CICR will hus modula e he a e and ex en o exocy osis
and esicle anspo o exocy ic si es.
We also used mi ochond ia as biosenso s o quan i y he local-
ized [Ca2+]c ansien s gene a ed du ing cell s imula ion. We ound
ha abou 50% o cell mi ochond ia become exposed o a cy osolic
Ca2+ concen a ion o 20–40
µM. These alues a e consis en wi h
he uppe epo ed es ima es o he local [Ca2+]c eached du ing
physiological s imula ion24. We canno es ima e he size o hese
ho spo s. Howe e , hal o he mi ochond ia in he same cells
de ec ed only 2–3
µM [Ca2+]c du ing s imula ion, indica ing he
p esence o a s eep subcellula [Ca2+]c g adien .
Finally, we specula e ha i ei he he loca ion o he Ca2+-
up ake p ope ies o hose mi ochond ia loca ed in he unc ional
uni s could be modula ed, his would be an e ec i e mechanism by
which o egula e sec e ion. I simila phenomena we e applicable
o neu ons, i would be also a e y simple mechanism by which o
gene a e synap ic plas ici y. Unde pa hological condi ions o du -
ing ageing, mi ochond ial damage may educe he abili y o mi o-
chond ia o ake up Ca2+. This would lead o inc eased sec e ion o
neu o ansmi e s and inc eased cell ac i a ion, a icious cycle ha
may igge p ocesses leading o nec osis o apop osis. h
Me hods
Cell cul u e and exp ession o aequo in.
Bo ine ad enal medulla ch oma in cells we e ob ained as desc ibed19 and used wi hin 1–3 days. The
mi ochond ial aequo in1,2 was a gi om T. Pozzan. Mu a ed (Asp 119 → Ala) mi ochond ial aequo in
was ob ained by eplacing, in- ame, complemen a y DNA encoding he wild- ype aequo in wi h ha
encoding he mu a ed aequo in15. Exp ession in ch oma in cells was achie ed by in ec ing he cells wi h
a de ec i e he pes simplex i us ype 1 con aining he mi ochond ial aequo in gene (pHSVmi AEQ).
Vi us packaging and i ing ha e been desc ibed25. Ch oma in-cell cul u es (5
×
105 cells pe 0.5
ml) we e
ou inely in ec ed wi h 2
×
103 in ec ious i us uni s 12–24 h be o e measu emen s. FCCP and CCCP we e
ob ained om Sigma. CGP37157 was om Toc is (B is ol, UK).
[Ca2+]M and [Ca2+]c measu emen s.
To econs i u e aequo ins, cells exp essing mi ochond ial aequo in we e incuba ed o 1–2
h a oom
empe a u e wi h 1
µM o ei he wild- ype coelen e azine o coelen e azine n, in s anda d medium
con aining 145
mM NaCl, 5 mM KCl, 1
mM
MgCl2, 1
mM CaCl2, 10
mM glucose, and 10
mM HEPES, pH
7.4. Cells we e hen placed in he pe usion chambe o a pu pose-buil he mos a ic luminome e a 22
°C o 37
°C. Fo expe imen s wi h pe meabilized cells, econs i u ion was pe o med as abo e and cells
we e placed in he luminome e . S anda d medium con aining 0.5 mM EGTA ins ead o CaCl2 was hen
pe used o 1 min, ollowed by a 1-min pe usion o in acellula medium (130
mM KCl, 10
mM NaCl,
1
mM MgCl2, 1
mM K3PO4, 0.2
mM EGTA, 1
mM ATP, 20
µM ADP, 2
mM succina e, 20
mM HEPES, pH
7) con aining 20
µM digi onin. In acellula medium wi hou digi onin was pe used o 1 min, ollowed
by bu e s o known [Ca2+], be ween 2 and 100
µM, p epa ed using HEDTA/Ca2+/Mg2+ mix u es.
Calib a ion was done using he calib a ion cu es co esponding o each aequo in ype. Calib a ion
cu es o AEQ3 a bo h 22
°C and 37
°C ha e been desc ibed16,17. Fo AEQ1 and AEQ2, new calib a ion
cu es a 37
°C we e gene a ed as desc ibed16. Aequo in consump ion was calcula ed as he in eg al o he
luminescence measu ed du ing he expe imen , no malized as a pe cen age. [Ca2+]c measu emen s we e
ob ained using Fu a-2 as desc ibed19. S a is ical alues a e gi en as means
±
s.e.m.
On-line measu emen s o ca echolamine elease.
Cells we e sc aped o ca e ully om he bo om o he Pe i dish wi h a ubbe policeman, and
cen i uged a 800
.p.m. (He aeus cen i uge) o 10
min. The cell pelle was esuspended in 200
µl K ebs-
HEPES medium (144 mM NaCl, 5.9 mM KCl, 2
mM CaCl2, 1.2
mM MgCl2, 11
mM glucose, 10
mM
HEPES, pH
7.4). Cells we e in oduced in a jacke ed mic ochambe o supe usion a 37
°C. The
supe usion a e was 2
ml
min–1. The liquid lowing om he supe usion chambe eached an
elec ochemical de ec o (model Me ohn AG CH-9100 He sau), placed jus a he ou le o he
mic ochambe , ha moni o s ‘on-line’, unde he ampe ome ic mode, he amoun o ca echolamines
sec e ed26. Cells we e s imula ed o sec e e wi h sho pulses (2
s) o K ebs-HEPES solu ion con aining
he s imuli.
RECEIVED 18 JUNE 1999; REVISED 1 NOVEMBER 1999; ACCEPTED 6 DECEMBER 1999;
PUBLISHED 20 DECEMBER 1999.
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ACKNOWLEDGEMENTS
We acknowledge inancial suppo om he Di ección Gene al de Enseñanza Supe io (g an PM98/
0142 o J.A. and g an PB97/0474 o J.G.-S.), om he Di ección Gene al de In es igación Cien í ica y
Técnica (g an PB94/0150) and om Janssen-Cilag o A.G.G., and om Jun a de Cas illa y León (g an
VA19/99 o J.A. and g an VA62/99 o M.T.A.). I.C.-I. and A.A. hold ellowships om he Minis e io de
Educación y Ciencia. We hank C. González and T. Pozzan o help ul commen s, and J. Fe nández o
echnical assis ance.
Co espondence and eques s o ma e ials should be add essed o J.A.